CN115364110B - Application of 6-phosphogluconic acid and derivatives thereof in preparation of medicines for preventing or treating sugar metabolic disorder diseases - Google Patents

Application of 6-phosphogluconic acid and derivatives thereof in preparation of medicines for preventing or treating sugar metabolic disorder diseases Download PDF

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CN115364110B
CN115364110B CN202211126387.6A CN202211126387A CN115364110B CN 115364110 B CN115364110 B CN 115364110B CN 202211126387 A CN202211126387 A CN 202211126387A CN 115364110 B CN115364110 B CN 115364110B
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phosphogluconate
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CN115364110A (en
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周璐
李晋
黄河
谢文豪
张裕丰
高玮
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Fudan University
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Abstract

The invention provides application of 6-phosphogluconic acid and derivatives thereof in preparing medicines for preventing or treating sugar metabolism disorder diseases, and the 6-phosphogluconic acid and derivatives thereof are cell sugar metabolism regulators and diabetes therapeutic agents with novel structures, particularly have remarkable regulation effect on islet alpha cells, and can be further prepared into medicines for preventing or treating sugar metabolism disorder diseases such as diabetes.

Description

Application of 6-phosphogluconic acid and derivatives thereof in preparation of medicines for preventing or treating sugar metabolic disorder diseases
Technical Field
The invention belongs to the field of pharmacy, and in particular relates to application of 6-phosphogluconic acid and derivatives thereof in preparing medicines for preventing or treating sugar metabolic disorder diseases.
Background
Diabetes is a syndrome of disturbed metabolism of sugar, fat, protein caused by absolute or relative deficiency of insulin secretion or impaired insulin utilization (i.e., insulin resistance), and is mainly marked by hyperglycemia, impaired sugar metabolism, diabetes. Acute complications during the course of the disease include diabetic ketoacidosis, hypertonic nonketotic diabetic coma, lactic acidosis caused by hypoglycemic treatment, and hypoglycemic coma. Chronic complications may be caused by long-term diabetic pathological conditions, which are mainly manifested by macrovascular lesions (heart disease, hypertension, cerebrovascular accident and lower limb vascular lesions), microangiopathy (diabetic retinopathy, diabetic nephropathy), inflammation (Endocr Relay cancer 2021.Doi: 10.1530/ERC-20-0315), neuropathy (Diabetes.2020.doi:10.2337/db19-0321.Diabetes Ther.2019.doi: 10.1007/s13300-019-00693-0.Diabetes Care.2003.doi: 10.2337/diacare.26.5.1553.), and the like. Meanwhile, studies have shown that diabetes is closely related to the occurrence of various malignant tumors (Endocr relay cancer 2009.Doi:10.1677/ERC-09-0087;Womens Health (Lond). 2011.Doi: 10.2217/whe.11.4.). The carcinogenic mechanisms of diabetes have proven to be complex, including excessive ROS production, destruction of basic biomolecules, chronic inflammation and impaired healing capacity, etc., which together lead to carcinogenesis in the pathological states of diabetes (cells.2020.doi: 10.3390/cells 9061380.). Typical examples are: pancreatic Cancer (Prim Care Diabetes.doi:10.1016/j.pcd.2018.11.015.Front Endocrinol Lausanne.doi: 10.3389/fendo.2020.563267.Ann Surg Oncol.doi:10.1245/s10434-014-3625-6)、 liver Cancer, stomach Cancer (Diabetes Res Clin practice. Doi: 10.1016/j. Diabores. 2022.109866), breast Cancer (Asian Pac J Cancer prev.2011; curr Cancer Drug targets. 2021), colorectal Cancer (Dig Dis sci.2012.Doi:10.1007/s10620-012-2055-1.Diabetes Technol Ther.doi: 10.1089/dia.2012.0263), biliary tract Cancer (Cancer cause controls. Doi:10.1007/s 10552-011-9754-3), urinary tract Cancer (Medicine Baltimore. Nov;96 (46): e 8588), head and neck Cancer (Acta diabetes. Doi:10.1007/s 00592-020-01643-0), and the like.
Diabetes mellitus is largely classified into type I, type II, other specific diabetes mellitus and gestational diabetes mellitus according to the cause of the disease and the pathological mechanism. Clinically, diabetes mellitus type I and diabetes mellitus type II are common. Type i diabetes, also known as insulin dependent diabetes, is thought to be caused by absolute deficiency of insulin secretion due to autoimmune destruction of beta cells in the islets. Type ii diabetes, also known as non-insulin dependent diabetes mellitus, is thought to be due to a deficiency in insulin receptor and a decrease in sensitivity, insulin resistance and a relative deficiency in insulin secretion characterized by a disorder in utilization. The reasons for these characteristics may be related to genetic environment, eating patterns, hormonal levels, and other metabolic disorders. Its typical symptoms are "three more and one less", i.e. polyuria, polydipsia, polyphagia and weight loss. According to the latest epidemiological study data, diabetes is one of the world's greatest epidemics. At the same time, the study predicts that by 2040 years the total number of adult patients worldwide is expected to exceed 6.5 billion. Because of the serious damage of diabetes to national life health and the wide variety of people suffering from the diabetes, the research of diabetes therapeutic drugs is always a hot spot and a difficult point in the field of drug research and development.
For type I diabetes, the clinical patients can only improve pathological symptoms by supplementing exogenous insulin or insulin analogues for a long time, and normal secretion of insulin cannot be restored fundamentally. In recent years, the use of immunomodulatory drugs to protect normal islet beta cells from autoimmune destruction has also been reported. However, immunotherapy is expensive, and complications and side effects of the treatment process are large. In addition, in the clinical practice of the front edge, cases of reconstructing the normal functions of pancreas of diabetics through pancreas transplantation operation exist, but the organ transplantation treatment has the defects of allograft rejection, lack of organ sources, complicated clinical technology and the like, and the treatment difficulty is extremely high. The main treatment mode of type II diabetes mellitus is the use of insulin and hypoglycemic agents, such as biguanides, sulfonylureas, thiazolidinediones, DPP-4 receptor inhibitors, SGLT-2 receptor inhibitors, GLP-1 analogues and the like. However, the use of insulin, or even abuse, can lead to progressively more severe insulin resistance, leading to progressive failure of insulin. Hypoglycemic agents can only improve hyperglycemia symptoms, cannot radically restore normal islet function, and patients need to take the hypoglycemic agents for life and have a plurality of obvious side effects.
The Pentose Phosphate Pathway (PPP) is another type of sugar metabolism pathway in organisms other than anaerobic glycolysis and aerobic oxidation of sugars, and can be divided into two stages. The first stage is an oxidation reaction, starting from the catalytic dehydrogenation of glucose-6-phosphate by glucose-6-phosphate dehydrogenase to 6-phosphogluconolactone, which hydrolyzes to 6-phosphogluconate under the action of lactonase, and then is dehydrogenated again and spontaneously decarboxylated to ribulose-5-phosphate under the action of glucose-6-phosphate dehydrogenase, with the simultaneous formation of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and CO 2. The second stage is a non-oxidative reaction, converting ribose to fructose-6-phosphate and glyceraldehyde-3-phosphate through a series of group transfer reactions into the glycolytic pathway, so the PPP pathway is also known as pentose phosphate bypass or hexose phosphate bypass. The PPP pathway has important biological significance: the large amounts of NADPH produced act as hydrogen donors to provide reducing agents for cellular metabolic reactions, such as participating in the synthesis of fatty acids and sterols, maintaining the reduced state of glutathione in erythrocytes; the abundant intermediate metabolites produced during the oxidation reaction stage provide raw materials for the synthesis of a variety of biomolecules (e.g., nucleotides, cholesterol, bile acids, steroid hormones, nonessential amino acids, erythrose 4-phosphate, etc.); the intermediate metabolite and the reaction regulating enzyme generated in the non-oxidative rearrangement stage are the same as most of the intermediate products and the reaction regulating enzymes of the calvin cycle in the photosynthesis of plants, so that the association of PPP and the photosynthesis can realize the interconversion between special monosaccharides; the PPP pathway is an independent oxidative decomposition of sugar initiated by direct oxidation of glucose, and is also a main pathway of pentose metabolism, so that the PPP pathway can complement the glycolytic pathway and tricarboxylic acid cycle, thereby enhancing the adaptive capacity of sugar metabolism of the organism. Ribose phosphate and NADPH provide the ribose skeleton of nucleotide synthesis in cell division proliferation and the electron donor of biosynthesis reactions. At the same time, PPP promotes glutamic acid generated by alpha-ketoglutarate, NADPH and NH 3, and then generates transamino reaction with other alpha-ketoacid to generate other amino acid, and the flux of the amino acid synthesis passage increases to promote cell division. This indicates that both the glycolytic pathway and the PPP metabolic pathway are of great importance in cell biosynthesis and division propagation.
Studies of scholars have found that artemether, an artemisinin derivative, can induce islet alpha cells to transform into islet beta cells, so that the normal insulin secretion function of islet tissues is recovered, and absolute insulin deficiency caused by islet beta cell deficiency is further reversed (cell.2017; doi: 10.1016/j.cell.2016.11.010). In addition, it has been reported that long-term administration of the endogenous metabolite gamma-aminobutyric acid (GABA) induces islet alpha cell mediated islet beta-like cell neogenesis in vivo, and the newly produced islet beta-like cells have complete islet beta cell function, and can replace native islet beta cells to partially restore the normal insulin secretion function of islet tissue in vivo (cell.2017; doi: 10.1016/j.cell.2016.11.002). The successful research cases of changing metabolic cell phenotype by using chemical molecules provide a new research strategy for fundamentally treating sugar metabolism disorder diseases, especially diabetes.
The 6-phosphogluconate is a key metabolic intermediate in the PPP pathway and is obtained by phosphorylating glucose by hexokinase, oxidative dehydrogenation of glucose-6-phosphate dehydrogenase and hydrolysis of 6-phosphogluconolactonase. Can be further oxidized and decarboxylated under the action of 6-phosphogluconate dehydrogenase to generate the ribulose-5-phosphate. The latter undergoes a series of transketosis/aldolization reactions, which are isomerized by metabolic intermediates such as tetrose phosphate, pentose phosphate, heptose phosphate and the like to produce glyceraldehyde-3-phosphate and fructose-6-phosphate which re-enter the glycolytic pathway. As a key metabolic intermediate in the sugar metabolism pathway, there are only a few reports of the decrease in 6-phosphogluconate level observed in erythrocytes of type II diabetics (j.clin. Med.2020; doi:10.3390/jcm 9061619), but since 6-phosphogluconate is too polar to penetrate the cell membrane into cells, there has been no study on its application as a regulator for the regulation of endogenous sugar metabolism pathway, nor on the further study on the chemical modification of the molecular structure of 6-phosphogluconate for the regulation of endogenous sugar metabolism pathway, nor on the application of 6-phosphogluconate and its similar compounds as a regulator of endogenous sugar metabolism pathway for the treatment of diabetes.
Disclosure of Invention
The first object of the present invention is to provide an application of 6-phosphogluconic acid and its derivatives, or pharmaceutically acceptable salts thereof, in preparing a medicament for preventing or treating a disorder of glucose metabolism, wherein the 6-phosphogluconic acid and its derivatives have a significant enhancement effect on insulin secretion function of islet alpha cells, and can be applied to treating the disorder of glucose metabolism as a regulator of endogenous glucose metabolism pathways.
The second object of the present invention is to provide a derivative of 6-phosphogluconate, wherein the derivative modified by 6-phosphogluconate can directly enter cells through cell membranes, and is absorbed by islet alpha cells to release 6-phosphogluconate, so that the expression of insulin secretion related genes in islet alpha cells is improved, and insulin secretion by islet alpha cells is stimulated.
The third object of the present invention is to provide a pharmaceutical composition, which comprises a pharmaceutically acceptable drug delivery vehicle for delivering 6-phosphogluconate or a derivative thereof or a pharmaceutically acceptable salt thereof into cells, and which is absorbed by islet alpha cells to increase the expression of insulin secretion-related genes in islet alpha cells, thereby stimulating insulin secretion by islet alpha cells.
A fourth object of the present invention is to provide an application of the pharmaceutical composition in preparing a medicament for preventing or treating a disorder of sugar metabolism including diabetes, ketoacidosis, hypertonic non-ketosis diabetic coma, diabetic retinopathy, diabetic nephropathy, diabetic foot, atherosclerosis, cerebrovascular accident, vascular lesions of lower limbs, obesity, fatty liver, malignant tumor, alzheimer's disease and parkinson's disease.
In order to achieve the first object, the invention provides application of 6-phosphogluconate and derivatives thereof, or pharmaceutically acceptable salts thereof in preparing medicines for preventing or treating sugar metabolism disorder diseases.
As a preferred embodiment, the 6-phosphogluconic acid derivative is a derivative with modified terminal phosphoric acid, including phosphate, phosphorane, phosphoylide, phosphate, phosphoramidate, phosphonate, phosphinate, phosphine oxide, thiophosphate, fluorophosphate, phosphoric anhydride, bisphosphonate, phosphite, phosphonite, thiophosphate, dithiophosphate, phosphoramidite;
Or derivatives modified with terminal carboxylic acids, including carboxylic esters, aminocarboxylic esters, amides, thiocarboxylic esters;
Or derivatives with modified hydroxyl groups on the sugar chain, including substituted hydroxyl groups, hydroxyl esters, hydroxyl amides, thiols, substituted thiols, thiol esters;
or a derivative in which each structural fragment is modified in combination in the above manner.
As a preferable scheme, the 6-phosphogluconic acid derivative has a structure shown in any one of the formulas (I), (II), (III), (IV) or (V):
In the method, in the process of the invention,
R 1-37 is selected from the group consisting of hydrogen atoms, alkyl and substituted alkyl, cycloalkyl and substituted cycloalkyl, alkylaryl and substituted alkylaryl groups of less than 10 carbon atoms, alkenyl and substituted alkenyl, cycloalkenyl and substituted cycloalkenyl, alkenylaryl and substituted alkenylaryl groups of less than 10 carbon atoms, alkynyl and substituted alkynyl, alkynylaryl and substituted alkynylaryl groups of less than 10 carbon atoms; or from biomolecules such as oligopeptides, glycosides, proteins, nucleotides, fatty acids, etc.; or a combination of the foregoing groups and fragments; the substituents are selected from halogen and heteroatoms.
As a preferred embodiment, the disorder of sugar metabolism includes diabetes, ketoacidosis, hypertonic non-ketosis diabetic coma, diabetic retinopathy, diabetic nephropathy, diabetic foot, atherosclerosis, cerebrovascular accident, lower limb vascular disease, obesity, fatty liver, malignant tumor, alzheimer's disease and Parkinson's disease.
In order to achieve the second object of the present invention, the present invention provides a derivative of 6-phosphogluconic acid, wherein the structure of the 6-phosphogluconic acid derivative is shown as any one of the formulas (I), (II), (III), (IV) or (V):
In the method, in the process of the invention,
R 1-37 is selected from the group consisting of hydrogen atoms, alkyl and substituted alkyl, cycloalkyl and substituted cycloalkyl, alkylaryl and substituted alkylaryl groups of less than 10 carbon atoms, alkenyl and substituted alkenyl, cycloalkenyl and substituted cycloalkenyl, alkenylaryl and substituted alkenylaryl groups of less than 10 carbon atoms, alkynyl and substituted alkynyl, alkynylaryl and substituted alkynylaryl groups of less than 10 carbon atoms; or from biomolecules such as oligopeptides, glycosides, proteins, nucleotides, fatty acids, etc.; or a combination of the foregoing groups and fragments; the substituents are selected from halogen and heteroatoms.
To achieve the third object of the present invention, the present invention provides a pharmaceutical composition comprising 6-phosphogluconate or a derivative thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable drug delivery vehicle, the pharmaceutical composition being permeable to a cell membrane after modification by the drug delivery vehicle.
As a preferred embodiment, the 6-phosphogluconic acid derivative is a derivative with modified terminal phosphoric acid, including phosphate, phosphorane, phosphoylide, phosphate, phosphoramidate, phosphonate, phosphinate, phosphine oxide, thiophosphate, fluorophosphate, phosphoric anhydride, bisphosphonate, phosphite, phosphonite, thiophosphate, dithiophosphate, phosphoramidite;
Or derivatives modified with terminal carboxylic acids, including carboxylic esters, aminocarboxylic esters, amides, thiocarboxylic esters;
Or derivatives with modified hydroxyl groups on the sugar chain, including substituted hydroxyl groups, hydroxyl esters, hydroxyl amides, thiols, substituted thiols, thiol esters;
or a derivative in which each structural fragment is modified in combination in the above manner.
As a preferable scheme, the 6-phosphogluconic acid derivative has a structure shown in any one of the formulas (I), (II), (III), (IV) or (V):
In the method, in the process of the invention,
R 1-37 is selected from the group consisting of hydrogen atoms, alkyl and substituted alkyl, cycloalkyl and substituted cycloalkyl, alkylaryl and substituted alkylaryl groups of less than 10 carbon atoms, alkenyl and substituted alkenyl, cycloalkenyl and substituted cycloalkenyl, alkenylaryl and substituted alkenylaryl groups of less than 10 carbon atoms, alkynyl and substituted alkynyl, alkynylaryl and substituted alkynylaryl groups of less than 10 carbon atoms; or from biomolecules such as oligopeptides, glycosides, proteins, nucleotides, fatty acids, etc.; or a combination of the foregoing groups and fragments; the substituents are selected from halogen and heteroatoms.
As a preferred scheme, the drug delivery carrier is a drug nano delivery system, comprising liposome, polymer micelle or microcapsule, lipid nanoparticle and chitosan nanoparticle, wherein the 6-phosphogluconic acid or derivative thereof, or pharmaceutically acceptable salt thereof is coated in the drug delivery carrier.
In order to achieve the fourth object of the present invention, the present invention provides the use of a pharmaceutical composition for the preparation of a medicament for preventing or treating a disorder of glucose metabolism including diabetes, ketoacidosis, hypertonic non-ketosis diabetic coma, diabetic retinopathy, diabetic nephropathy, diabetic foot, atherosclerosis, cerebrovascular accident, lower limb vascular disease, obesity, fatty liver, malignant tumor, alzheimer's disease and Parkinson's disease.
Experiments prove that the preparation of 6-phosphogluconate, 6-phosphogluconate and/or phosphate derivatives or the preparation of 6-phosphogluconate, 6-phosphogluconate and/or phosphate salts or the preparation of 6-phosphogluconate modified by the drug delivery carrier can improve the membrane permeability of the preparation, enter cells through cell membranes, are absorbed by islet alpha cells and release 6-phosphogluconate, improve the expression of insulin secretion related genes in the islet alpha cells, and stimulate the islet alpha cells to secrete insulin.
The drug delivery carrier is a nano delivery system of a drug composite (combination) solution or a suspension, and the 6-phosphogluconic acid or the derivative thereof or the pharmaceutically acceptable salt thereof is coated in the nano delivery system of the drug composite (combination) solution or the suspension.
The invention is based on the finding that human islet alpha cells have significantly lower 6-phosphogluconate content than beta cells through metabonomic studies. After exogenous supplementation of 6-phosphogluconate or derivatives thereof, islet alpha cells show high insulin expression level, and q-PCR detection shows that the transcription level of insulin synthesis related genes in islet alpha cells treated by the 6-phosphogluconate or derivatives thereof is obviously increased, and islet beta cell-like functionalization is shown. The 6-phosphogluconate itself has large polar groups (carboxylate, phosphate, etc.), and cannot permeate the cell membrane and be absorbed by islet alpha cells. Therefore, 6-phosphogluconic acid or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable drug delivery carrier are prepared into a composition capable of penetrating through cell membranes, or the chemical structure of the 6-phosphogluconic acid is modified, the purpose of increasing the concentration of 6-phosphogluconic acid in cells is achieved by taking 6-phosphogluconic acid derivatives as 6-phosphogluconic acid precursors, the 6-phosphogluconic acid can directly penetrate through the cell membranes to enter the cells, and can be absorbed and released by islet alpha cells, so that the expression of insulin secretion related genes in the islet alpha cells is improved, and insulin secretion of the islet alpha cells is stimulated.
In the examples of the present invention, it is preferable to prepare derivatives of the following structural formula and verify the biological properties, and the 6-phosphogluconate and/or phosphate derivatives can be absorbed by cells and release 6-phosphogluconate.
The scope of the present invention is not intended to be exhaustive synthesis of all 6-phosphogluconic acids as prodrugs of derivatives of carboxylic esters, phosphoric esters, hydroxy esters, amides, phosphoramides, phosphorothioates, carboxylic esters, aminocarboxylic esters, amides, thiocarboxylic esters, hydroxy esters, hydroxyamides, thiols, substituted thiols, thiolesters, phosphonates, mono/polyphosphonates, phosphoranes, phosphotides, phosphates, phosphoramidates, phosphinates, phosphine oxides, thiophosphates, fluorophosphates, phosphoric anhydrides, bisphosphates, phosphites, phosphonites, phosphorothioates, phosphorodithioates, phosphoramidites, halo-substituted derivatives, possible modification strategies related to the functional group of 6-phosphogluconic acid molecule in the prior art may be used as potential modification methods, the 6-phosphogluconate derivatives are of course regarded as derivatives in which each structural fragment is modified by a combination of the above carboxylic acid esters, phosphoric acid esters, hydroxy esters, amides, phosphoramides, phosphorothioates, carboxylic acid esters, aminocarboxylic acid esters, amides, thiocarboxylic acid esters, hydroxy groups, hydroxy esters, hydroxyamides, thiols, substituted thiols, thiolates, phosphonates, mono/polyphosphonates, phosphoranes, phosphotides, phosphates, phosphoramidates, phosphinates, phosphine oxides, thiophosphates, fluorophosphates, phosphoric anhydrides, bisphosphonates, phosphites, phosphonites, phosphorothioates, phosphorodithioates, phosphoramides, halogenated and the like resulting from modification of each structural fragment by the above.
Pharmaceutically acceptable salts in the invention are sodium salt, potassium salt, calcium salt, ammonium salt, amino acid salt and other salts suitable for pharmaceutical use.
Some of the compounds of the present invention contain acidic groups which form salts with bases and can form salts of derivatives by conventional means, including sodium, potassium, calcium, ammonium salts or with amino acids such as lysine, arginine, histidine and the like, with preferred salts being sodium and potassium salts. Similarly, the compounds of the invention contain basic groups which form salts with acids and salts of derivatives may be formed by conventional means, including organic acid salts such as acetates, citrates, fumarates, maleates, oxalates, malates, citrates, succinates, tartrates, lactates, camphorsulfonates, benzenesulfonates, p-toluenesulfonates, methanesulfonates, trifluoroacetates, trifluoromethanesulfonates and the like; inorganic acid salts such as hydrohalic acid (hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid) salts, sulfates, phosphates, nitrates, and the like. Or with amino acids such as glutamic acid or aspartic acid to form glutamate or aspartate, preferred salts being hydrochloride, bromhydrochloride.
The term "halogen" refers to fluorine, chlorine, bromine and iodine, preferably fluorine and chlorine.
The term "alkyl" refers to saturated aliphatic groups, including straight chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like), branched chain alkyl groups (isopropyl, tert-butyl, isobutyl, and the like), cycloalkyl groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl), alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups, as well as other group-substituted alkyl and cycloalkyl groups. In certain embodiments, the linear or branched alkyl groups have 4 or fewer carbon atoms in the backbone.
The term "alkenyl" refers to an unsaturated aliphatic group having a carbon-carbon double bond, and includes straight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched chain alkenyl groups (isopropenyl, isobutenyl, etc.), cycloalkenyl groups (cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, etc.), alkenyl-substituted cycloalkenyl groups, and cycloalkenyl-substituted alkenyl groups, as well as other group-substituted alkenyl and cycloalkenyl groups. In certain embodiments, the linear or branched alkenyl group has 4 or fewer carbon atoms in the backbone.
The term "alkynyl" refers to an unsaturated aliphatic group bearing a carbon-carbon triple bond, and includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, and the like), branched-chain alkynyl groups (isopentynyl, isohexynyl, and the like), and alkynyl groups substituted with other groups. In certain embodiments, straight or branched chain alkynyl groups have 4 or fewer carbon atoms in the backbone.
Pharmaceutically acceptable drug delivery vehicles in the present invention mean that 6-phosphogluconate and its derivatives can be assisted by being absorbed by islet alpha cells by being combined with or entrapped by 6-phosphogluconate and its derivatives, which cross the cell membrane. The delivery system is a nano delivery system of a drug composite (composition) solution or suspension, and comprises liposome, polymer micelle or microcapsule, lipid nanoparticle and the like, wherein 6-phosphogluconic acid or derivatives thereof or pharmaceutically acceptable salts thereof are coated in the composite or nano carrier.
The pharmaceutical composition of the present invention may further contain one or more pharmaceutically acceptable carriers other than pharmaceutically acceptable drug delivery carriers, including diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and the like which are conventional in the pharmaceutical field, and flavoring agents, sweeteners and the like may be added as necessary.
The pharmaceutical compositions of the invention may be prepared in any form, for example granules, powders, tablets, coated tablets, capsules, pills, syrups, drops, solutions, suspensions and emulsions, or sustained release formulations of the active ingredient, wherein the capsules include hard or soft gelatin capsules, and the granules and powders may be in non-effervescent or effervescent form.
The pharmaceutical compositions of the present invention may be administered according to conventional methods by a variety of routes including oral, intravenous, intra-arterial, intraperitoneal, intrathoracic, transdermal, nasal, inhalation, rectal, ocular and subcutaneous introduction.
The invention adopts a quantitative real-time polymerase chain reaction (q-PCR) fluorescence quantitative method reported in the literature to measure the influence degree of 6-phosphogluconic acid and derivatives thereof (including pharmaceutically acceptable salts thereof) as metabolic regulators on the improvement of the transcription level of insulin synthesis related genes in islet alpha cells in vitro. The q-PCR is mainly to monitor the change of the amplification product quantity of each cycle in the PCR amplification reaction in real time through the change of fluorescent signals, and the common fluorescent labeling method comprises SYBR green I dye and TaqMan probe. And finally, quantitatively analyzing the initial template through the relation between the Ct value and the standard curve, and calculating the improvement degree of the metabolic regulator on the transcription level of the insulin synthesis related genes in the islet alpha cells.
Experiments prove that the disclosed compounds are brand-new cell glucose metabolism regulators and diabetes therapeutic agents, and particularly have remarkable regulation effects on islet alpha cells. And because of being derived from endogenous metabolites, the potential toxic and side effects are extremely low. Can be further prepared into medicines for preventing or treating diabetes and other sugar metabolism disorder diseases, including diabetes related complications such as ketoacidosis, hypertonic non-ketosis diabetic coma, diabetic retinopathy, diabetic nephropathy, and diabetic foot; cardiovascular and cerebrovascular related diseases such as heart and macrovascular microangiopathy, cardiomyopathy, coronary heart disease, cerebral arteriosclerosis, ischemic cerebrovascular disease, etc.; diseases of the central system, such as neurodegenerative diseases like Alzheimer's disease, parkinson's disease, etc.
The invention has the advantages that the 6-phosphogluconic acid and the derivatives thereof (including the carboxyl/phosphate ester compounds derived from the 6-phosphogluconic acid and the derivatives thereof as prodrugs) are cell glucose metabolism modulators and diabetes therapeutic agents with novel structures, particularly have remarkable regulating effect on islet alpha cells, and can be further prepared into medicaments for preventing or treating diabetes and other glucose metabolism disorder diseases.
Drawings
FIG. 1. Metabolite differences between islet alpha cells and beta cells;
FIG. 2 LC-MS/MS experiments targeting detection of 6-phosphogluconate;
FIG. 3 shows that the target compound has significantly increased transcription level of insulin synthesis-related genes in islet alpha cells at a concentration of 10. Mu.M;
FIG. 4.6 verification of the effect of methyl-6-phosphogluconate on islet beta cell regeneration;
FIG. 5. Immunofluorescence assay to measure islet beta cell regeneration in type I diabetic mice;
FIG. 6A hypersensitivity insulin kit for measuring insulin concentration in peripheral blood of mice.
Detailed Description
Hereinafter, the technology of the present invention will be described in detail with reference to the specific embodiments. It should be understood that the following detailed description is merely intended to aid those skilled in the art in understanding the invention, and is not intended to limit the invention.
EXAMPLE 1 islet alpha cell and beta cell metabolite concentration determination and comparison
1) Metabonomics detection of islet alpha cells and beta cells
1X 10 6 of alpha cells and beta cells were placed in a 15mL centrifuge tube, and 4.5mL of methanol at-80℃was added: after mixing the solutions in water (v: v=4:1), incubation was carried out for 20min at-80 ℃. After the centrifuge tube was removed and vortexed for 1min with shaking, the tube was centrifuged at 14,000Xg for 5min, and the supernatant was placed in a fresh centrifuge tube. The supernatant was dried by vacuum centrifugal drying or nitrogen blowing. The dried metabolites were reconstituted with 100 μl of 35% acetonitrile solution and the metabolome in α and β cells was detected using LC-MS/MS targeted metabolomics. The heat map shows metabonomics results that show significant differences in metabolites of islet alpha cells versus beta cells (fig. 1).
2) Detection of islet alpha and beta cell 6-phosphogluconate
The 6-phosphogluconate in the alpha cells and the beta cells is extracted according to the above-mentioned method for extracting the metabolites. The dried metabolite was reconstituted with 100. Mu.L of 35% acetonitrile and targeted detection of 6-phosphogluconic acid was performed by LC-MS/MS method. MRM: m/z 275- >79, dp= -93V, ce= -10V. LC-MS/MS experiments showed that 6-phosphogluconate was relatively high in beta cells (fig. 2).
EXAMPLE 2 preparation of derivative of 6-phosphogluconate methyl 6-phosphogluconate (formula 1-1)
The compound shown in the general formula (1) is obtained by forming ester with corresponding alcohol by using 6-phosphogluconic acid under the acid catalysis, wherein the reaction condition generally adopts alcohol as a solvent, and HCl gas is introduced as a catalyst. And 6-phosphogluconate is a known compound, and can be obtained by phosphorylating and oxidizing glucose. The following reaction equations and synthesis conditions detail the synthesis of formula (1).
1) Synthesis of glucose-6-phosphate
2) Synthesis of 6-phosphogluconate
3) Synthesis of methyl 6-phosphogluconate (Compound 1-1) (Synthesis method one)
1G (0.0029 mol,1.0 eq) of Compound 3 was weighed into a 50mL three-necked flask, 25mL of anhydrous methanol was added for dissolution, and the reaction solution was stirred with dry hydrogen chloride gas introduced thereinto, stirred overnight, and the reaction was detected by LC-MS. Concentrating under reduced pressure to obtain oily crude product, dissolving with HPLC grade methanol, and purifying by HPLC to obtain white solid 0.7g, namely methyl 6-phosphogluconate (formula 1-1), yield 82％.MS(ESI)(m/z)： 289[M-H]-.1H NMR(400MHz,DMSO-d6)δ5.18(s,1H),4.51(s,1H),4.37 (s,2H),4.33(m,1H),4.29(m,2H),4.2(s,2H),4.13(d,1H),3.70(s,3H),3.50(m,1H),3.40(m,1H).
EXAMPLE 3 preparation of Compound 1-2、1-3、2-1、2-2、2-3、3-1、3-2、3-3、4-1、4-2、 4-3、5-1、5-2、5-3、6-1、6-2、6-3、7-1、7-2、7-3、8-1、8-2、8-3、9-1、 9-2、9-3、10-1、10-2、10-3、11-1、11-2、11-3、12-1、12-2、12-3、13-1、 13-2、13-3、14-1、14-2、14-3、15-1、15-2、15-3、16-1、16-2、16-3、17-1、 17-2、17-3、18-1、18-2、18-3、19-1、19-2、19-3、20-1、20-2、20-3、21-1、 21-2、21-3、22-1、22-2、22-3、23-1、23-2、23-3、24-1、24-2、24-3、25-1、 25-2、25-3
Referring to the conditions and steps of the first synthetic method in example 2, dimethyl 6-phosphogluconate (formula 1-2) was obtained in a yield of 20% by reacting anhydrous methanol with compound 3.
Referring to the conditions and steps of the first synthetic method in example 2, trimethyl 6-phosphogluconate (formula 1-3) was prepared by reacting anhydrous methanol with compound 3 in a yield of 10%.
Referring to the conditions and steps of the first synthetic method in example 2, ethyl 6-phosphogluconate (formula 2-1) was obtained in a yield of 70% by reacting absolute ethanol with compound 3.
Referring to the conditions and steps of the first synthetic method in example 2, diethyl 6-phosphogluconate (formula 2-2) was obtained by reacting absolute ethanol with compound 3 in a yield of 20%.
Referring to the conditions and steps of the first synthetic method in example 2, triethyl 6-phosphogluconate (formula 2-3) was prepared by reacting absolute ethanol with compound 3 in a yield of 10%.
With reference to the conditions and steps of the first synthetic method in example 2, propyl 6-phosphogluconate (formula 3-1) was obtained in a yield of 70% by reacting anhydrous propanol with compound 3.
With reference to the conditions and steps of the first synthetic method in example 2, dipropyl 6-phosphogluconate (formula 3-2) was obtained in a yield of 20% by reacting anhydrous propanol with compound 3.
With reference to the conditions and steps of the first synthetic method in example 2, tripropyl 6-phosphogluconate (formula 3-3) was obtained in a yield of 10% by reacting anhydrous propanol with compound 3.
Referring to the conditions and steps of the first synthetic method in example 2, isopropyl 6-phosphogluconate (formula 4-1) was obtained in a yield of 70% by reacting anhydrous isopropyl alcohol with compound 3.
Referring to the conditions and steps of the first synthetic method in example 2, anhydrous isopropanol was reacted with compound 3 to obtain diisopropyl 6-phosphogluconate (formula 4-2) in a yield of 20%.
Referring to the conditions and steps of the first synthetic method in example 2, triisopropanol 6-phosphogluconate (formula 4-3) was prepared by reacting anhydrous isopropanol with compound 3 in a yield of 10%.
With reference to the conditions and steps of the first synthetic method in example 2, butyl 6-phosphogluconate (formula 5-1) was obtained in a yield of 70% by reacting dry butanol with compound 3.
With reference to the conditions and steps of the first synthetic method in example 2, dibutyl 6-phosphogluconate (formula 5-2) was obtained in a yield of 20% by reacting dry butanol with compound 3.
With reference to the conditions and steps of the first synthetic method in example 2, tributyl 6-phosphogluconate (formula 5-3) was obtained in a yield of 10% by reacting dry butanol with compound 3.
With reference to the conditions and steps of the first synthetic method in example 2, sec-butyl 6-phosphogluconate (formula 6-1) was obtained in a yield of 70% by reacting anhydrous sec-butanol with compound 3.
With reference to the conditions and steps of the first synthetic method in example 2, di-sec-butyl 6-phosphogluconate (formula 6-2) was prepared by reacting anhydrous sec-butanol with compound 3 in a yield of 20%.
With reference to the conditions and steps of the first synthetic method in example 2, the tri-sec-butyl 6-phosphogluconate (formula 6-3) was obtained by reacting anhydrous sec-butanol with compound 3 in a yield of 10%.
Referring to the conditions and steps of the first synthetic method in example 2, isobutyl 6-phosphogluconate (formula 7-1) was obtained in a yield of 70% by reacting dry isobutanol with compound 3.
With reference to the conditions and steps of the first synthetic method in example 2, diisobutyl 6-phosphogluconate (formula 7-2) was obtained in a yield of 20% by reacting dry isobutanol with compound 3.
By referring to the conditions and steps of the first synthetic method in example 2, triisobutyl 6-phosphogluconate (formula 7-3) was obtained in a yield of 10% by reacting dry isobutanol with compound 3.
With reference to the conditions and steps of the first synthetic method in example 2, tert-butyl 6-phosphogluconate (formula 8-1) was obtained in a yield of 70% by reacting anhydrous tert-butanol with compound 3.
With reference to the conditions and steps of the first synthetic method in example 2, di-tert-butyl 6-phosphogluconate (formula 8-2) was obtained in a yield of 20% by reacting anhydrous tert-butanol with compound 3.
With reference to the conditions and steps of the first synthetic method in example 2, the tri-tert-butyl 6-phosphogluconate (formula 8-3) was obtained by reacting anhydrous tert-butanol with compound 3 in a yield of 10%.
Referring to the conditions and steps of the first synthetic method in example 2, amyl alcohol was reacted with compound 3 to produce amyl 6-phosphogluconate (formula 9-1) in a yield of 70%.
With reference to the conditions and steps of the first synthetic method in example 2, dipentyl 6-phosphogluconate (formula 9-2) was obtained by reacting anhydrous amyl alcohol with compound 3 in a yield of 20%.
With reference to the conditions and steps of the first synthetic method in example 2, the reaction of anhydrous amyl alcohol with compound 3 produced tripentyl 6-phosphogluconate (formula 9-3) in a yield of 10%.
With reference to the conditions and steps of the first synthetic method in example 2, secondary amyl alcohol 6-phosphogluconate (formula 10-1) is prepared by reacting anhydrous secondary amyl alcohol with a compound 3, and the yield is 70%.
With reference to the conditions and steps of the first synthetic method in example 2, the anhydrous secondary amyl alcohol and the compound 3 are reacted to prepare the secondary amyl 6-phosphogluconate (formula 10-2), and the yield is 20%.
Referring to the conditions and steps of the first synthetic method in example 2, the anhydrous sec-amyl alcohol and the compound 3 are reacted to prepare the 6-phosphogluconate tri-sec-amyl alcohol (formula 10-3), and the yield is 10%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2-methyl butanol and compound 3 were reacted to obtain 6-phosphogluconate-2-methyl butanol ester (formula 11-1) in a yield of 70%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2-methyl butanol and compound 3 were reacted to obtain 2-methyl-2-butanolate 6-phosphogluconate (formula 11-2) in a yield of 20%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2-methyl butanol and compound 3 were reacted to obtain 6-phosphogluconate-2-methyltributanol ester (formula 11-3) in a yield of 10%.
Referring to the conditions and steps of the first synthetic method in example 2, isoamyl 6-phosphogluconate (formula 12-1) was prepared by reacting anhydrous isoamyl alcohol with compound 3 in a yield of 70%.
Referring to the conditions and steps of the first synthetic method in example 2, diisoamyl alcohol 6-phosphogluconate (formula 12-2) was prepared by reacting anhydrous isoamyl alcohol with compound 3 in a yield of 20%.
Referring to the conditions and steps of the first synthetic method in example 2, triisoamyl alcohol 6-phosphogluconate (formula 12-3) was obtained in a yield of 10% by reacting anhydrous isoamyl alcohol with compound 3.
Referring to the conditions and steps of the first synthetic method in example 2, neopentyl alcohol is reacted with compound 3 to prepare neopentyl alcohol 6-phosphogluconate (formula 13-1) with a yield of 70%.
Referring to the conditions and steps of the first synthetic method in example 2, the anhydrous neopentyl alcohol is reacted with the compound 3 to prepare the 6-phosphogluconate di-neopentyl alcohol ester (formula 13-2) with the yield of 20%.
Referring to the conditions and steps of the first synthetic method in example 2, the anhydrous neopentyl alcohol is reacted with the compound 3 to prepare the 6-phosphogluconate trisneopentyl alcohol ester (formula 13-3), and the yield is 10%.
With reference to the conditions and steps of the first synthetic method in example 2, the reaction of anhydrous hexanol with compound 3 gave hexanol 6-phosphogluconate (formula 14-1) in a yield of 70%.
Referring to the conditions and steps of the first synthetic method in example 2, dihexyl 6-phosphogluconate (formula 14-2) was obtained in a yield of 20% by reacting anhydrous hexanol with compound 3.
Referring to the conditions and steps of the first synthetic method in example 2, the reaction of anhydrous hexanol with compound 3 gave a trienol 6-phosphogluconate (formula 14-3) in a yield of 10%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2-ethylhexanol and compound 3 were reacted to obtain 2-ethylhexanol 6-phosphogluconate (formula 15-1) in a yield of 70%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2-ethylhexanol and compound 3 were reacted to obtain 2-ethyldihexyl 6-phosphogluconate (formula 15-2) in a yield of 20%.
With reference to the conditions and steps of the first synthetic method in example 2, 2-ethylhexanol phosphate, 2-ethylcyclohexanol 6-phosphogluconate (formula 15-3) was obtained by reacting anhydrous 2-ethylhexanol with compound 3 in a yield of 10%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 1, 3-dimethylbutanol and compound 3 are reacted to obtain 6-phosphogluconate-1, 3-dimethylbutanol ester (formula 16-1) in a yield of 70%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 1, 3-dimethylbutanol and compound 3 are reacted to obtain 6-phosphogluconate-1, 3-dimethylbutanol ester (formula 16-2) in a yield of 20%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 1, 3-dimethylbutanol and compound 3 are reacted to obtain 6-phosphogluconate-1, 3-dimethylbutanol ester (formula 16-3) in a yield of 10%.
With reference to the conditions and steps of the first synthetic method in example 2, the reaction of anhydrous heptanol with compound 3 produced heptylphospholate 6 (formula 17-1) in a yield of 70%.
With reference to the conditions and steps of the first synthetic method in example 2, diheptyl 6-phosphogluconate (formula 17-2) was obtained in a yield of 20% by reacting anhydrous heptanol with compound 3.
With reference to the conditions and steps of the first synthetic method in example 2, the reaction of anhydrous heptanol with compound 3 gives tri-heptyl 6-phosphogluconate (formula 17-3) in a yield of 10%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 1-methylheptanol was reacted with compound 3 to obtain glucono-1-methylheptanol 6-phosphate (formula 18-1) in a yield of 70%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 1-methylheptanol was reacted with compound 3 to obtain glucono-1-methyldiheptanol 6-phosphate (formula 18-2) in a yield of 20%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 1-methylheptanol was reacted with compound 3 to obtain glucono-1-methyltrispanol 6-phosphate (formula 18-3) in a yield of 10%.
Referring to the conditions and steps of the first synthetic method in example 2, anhydrous 3-methyl-3-buten-1-ol was reacted with compound 3 to obtain 6-phosphogluconate-3-methyl-3-buten-1-ol ester (formula 19-1) in a yield of 70%.
Referring to the conditions and steps of the first synthetic method in example 2, anhydrous 3-methyl-3-buten-1-ol was reacted with compound 3 to obtain 6-phosphogluconate-3-methyl-3-buten-1-ol diester (formula 19-2) in a yield of 20%.
Referring to the conditions and steps of the first synthetic method in example 2, anhydrous 3-methyl-3-buten-1-ol was reacted with compound 3 to obtain 6-phosphogluconate-3-methyl-3-buten-1-ol triester (formula 19-3) in a yield of 10%.
Referring to the conditions and steps of the first synthetic method in example 2, benzyl alcohol 6-phosphogluconate (formula 20-1) was obtained by reacting anhydrous benzyl alcohol with compound 3 in a yield of 70%.
Referring to the conditions and steps of the first synthetic method in example 2, benzyl alcohol 6-phosphogluconate is prepared by reacting anhydrous benzyl alcohol with a compound 3 (formula 20-2), and the yield is 20%.
Referring to the conditions and steps of the first synthetic method in example 2, benzyl alcohol anhydride was reacted with compound 3 to obtain benzyl 6-phosphogluconate triester (formula 20-3) in a yield of 10%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2, 6-diisopropylphenol is reacted with compound 3 to produce 6-phosphogluconate-2, 6-diisopropylphenol ester (formula 21-1) in a yield of 70%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2, 6-diisopropylphenol is reacted with compound 3 to produce 6-phosphogluconate-2, 6-diisopropylphenol diester (formula 21-2) in a yield of 20%.
With reference to the conditions and steps of the first synthetic procedure in example 2, anhydrous 2, 6-diisopropylphenol is reacted with compound 3 to produce 6-phosphogluconate-2, 6-diisopropylphenol triester (formula 21-3) in a yield of 10%.
Referring to the conditions and steps of the first synthetic method in example 2, cyclohexylmethyl 6-phosphogluconate (formula 22-1) was obtained in a yield of 70% by reacting anhydrous cyclohexylmethanol with compound 3.
Referring to the conditions and steps of the first synthetic method in example 2, cyclohexylmethyl 6-phosphogluconate (formula 22-2) was obtained in a yield of 20% by reacting anhydrous cyclohexylmethanol with compound 3.
Referring to the conditions and steps of the first synthetic method in example 2, cyclohexylmethyl 6-phosphogluconate (formula 22-3) was obtained in a yield of 10% by reacting anhydrous cyclohexylmethanol with compound 3.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2-morpholinoethanol and compound 3 are reacted to obtain 6-phosphogluconate-2-morpholinoethyl (formula 23-1) in a yield of 70%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2-morpholinoethanol was reacted with compound 3 to obtain 2-morpholinoethyl 6-phosphogluconate (formula 23-2) in a yield of 20%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2-morpholinoethanol and compound 3 are reacted to obtain 6-phosphogluconate-2-morpholinoethyl ester (formula 23-3) in a yield of 10%.
Referring to the conditions and steps of the first synthetic method in example 2, cyclohexylethyl 6-phosphogluconate (formula 24-1) was obtained in a yield of 70% by reacting anhydrous cyclohexylethanol with compound 3.
Referring to the conditions and steps of the first synthetic method in example 2, anhydrous cyclohexylethanol was reacted with compound 3 to obtain cyclohexyldiethyl 6-phosphogluconate (formula 24-2), in a yield of 20%.
Referring to the conditions and steps of the first synthetic method in example 2, cyclohexyltriethyl 6-phosphate (formula 24-3) was produced by reacting anhydrous cyclohexylethanol with compound 3 in a yield of 10%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2-fluoroethanol was reacted with compound 3 to obtain 6-phosphogluconate-2-fluoroethyl ester (formula 25-1) in a yield of 70%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2-fluoroethanol was reacted with compound 3 to obtain 2-fluorodiethyl 6-phosphogluconate (formula 25-2) in a yield of 20%.
With reference to the conditions and steps of the first synthetic method in example 2, anhydrous 2-fluoroethanol and compound 3 are reacted to obtain 6-phosphogluconate-2-fluorotriethyl ester (formula 25-3) in a yield of 10%.
EXAMPLE 4 preparation of N-ethyl-6-phosphoglucosamide (formula 26-1)
1) Synthesis of N-ethyl-6-phosphoglucosamide (formula 26-1) (Synthesis method II)
1G of Compound 3 (0.0029 mol,1.0 eq) was weighed out and dissolved well in 10mL of methanol in a 50mL round bottom flask. 0.47g of ethylamine hydrochloride (0.006mol, 2.0 eq), 1.47g of iodine (0.006 mol,2.0 eq) and 0.83g of K 2CO3 (0.006mol, 2.0 eq) were weighed into a methanol solution system of compound 3 with stirring. The reaction was stirred at room temperature for 12h and monitored by lc-MS in real time until complete consumption of compound 3. The reaction solution was filtered and concentrated by distillation under reduced pressure to give a crude yellow product, which was then purified by separation using a resin column (Dowex 8WX-100, acid form) to give 0.65g of a white solid in the yield of 74％,MS(ESI) (m/z)：302[M-H]-.1H NMR(400MHz,DMSO-d6)δ8.01(s,1H),5.18(s, 1H),4.51(s,1H),4.44(d,1H),4.37(s,2H),4.29(m,2H),4.20(s,2H),3.80(m,1H),3.50(m,1H),3.40(m,1H),3.24(m,2H),0.99(m,3H).
EXAMPLE 5 preparation of Compounds 26-2, 26-3, 27-1, 27-2, 27-3, 28-1, 28-2, 28-3
Referring to the conditions and steps of the synthesis method II in example 4, di-N-ethyl-6-phosphoglucosamide (formula 26-2) was prepared by reacting anhydrous ethylamine hydrochloride with compound 3 in a yield of 20%.
Referring to the conditions and steps of the synthesis method II in example 4, tri-N-ethyl-6-phosphoglucosamide (formula 26-3) was prepared by reacting anhydrous ethylamine hydrochloride with compound 3 in a yield of 10%.
Referring to the conditions and steps of the synthesis method II in example 4, N-2-ethyl-6-phosphoglucosamide (formula 27-1) was prepared by reacting anhydrous diethylamine with compound 3 in a yield of 70%.
With reference to the conditions and steps of the synthesis method II in example 4, di-N, N-2-ethyl-6-phosphoglucosamide (formula 27-2) was prepared by reacting anhydrous diethylamine with compound 3 in a yield of 20%.
Referring to the conditions and steps of the synthesis method II in example 4, tri-N, N-2-ethyl-6-phosphoglucosamide (formula 27-3) was prepared by reacting anhydrous diethylamine with compound 3 in a yield of 10%.
By referring to the conditions and steps of the synthesis method II in example 4, anhydrous p-bromophenyl-6-phosphoglucosamide (formula 28-1) was obtained by reacting compound 3 with an anhydrous p-bromophenyl amine, and the yield was 70%.
With reference to the conditions and steps of the synthesis method II in example 4, di-N-p-bromophenyl-6-phosphoglucosamide (formula 28-2) was prepared by reacting anhydrous p-bromoaniline with compound 3 in a yield of 20%.
Referring to the conditions and steps of the synthesis method II in example 4, tri-N-p-bromophenyl-6-phosphoglucosamide (formula 28-3) was prepared by reacting anhydrous p-bromophenyl amine with compound 3 in a yield of 10%.
Example 6.6 in vitro determination of the Effect of Glucononic acid 6-phosphate and its derivatives (including pharmaceutically acceptable salts derived therefrom) on the increase in the transcriptional level of insulin synthesis-related genes in islet alpha cells
Experimental apparatus and materials
1.Q-PCR test kit (Shanghai Biyun biotechnology Co., ltd.)
Quant Studio 3 real-time fluorescent quantitative PCR System (Semer Feishier technology (China) Co., ltd.)
4.Q-PCR target nucleic acid sequence primer (Beijing qingke biotechnology Co., ltd.)
3. Electric heating thermostatic water bath (Shanghai-constant technology Co., ltd.).
4. Vortex mixer (Shanghai precision Co., ltd. Model XW-80A).
5. High speed centrifuge (model Eppendorf 5804R).
6. The organic solvents used were purchased from Shanghai national pharmaceutical reagent company, methanol was chromatographic pure, water was filtered by Milli-Q pump, deionized, ultra-pure water ultrafiltered by 0.22 μm membrane, and other biological consumables were purchased from domestic company.
7. Preparing a compound solution to be tested: 1-2mg of each compound to be tested is accurately weighed, and a proper amount of PBS is firstly added to accurately prepare a 10mmol/L stock solution. Taking a certain volume of PBS stock solution of the compound to be tested, and adding a proper volume of PBS to dilute the compound to be tested to a solution with a required concentration.
The in vitro effect of 6-phosphogluconate and derivatives thereof (including pharmaceutically acceptable salts derived therefrom) on the increase in the transcriptional level of genes involved in insulin synthesis in islet alpha cells is measured.
Sample preparation: islet alpha cells after 72h of administration were taken, added with Trizol, and left at room temperature for 5min to allow sufficient lysis. After completion of the lysis, the lysate was centrifuged at 12000rmp at 4℃for 5min, and the supernatant was collected. Chloroform (200 ul/mL Trizol) was added to the supernatant, and the mixture was gently shaken for 30 seconds and then left at room temperature for 15 minutes. After that, the mixture was centrifuged at 12000rmp at 4℃for 15min. Sucking the supernatant part into another centrifuge tube, adding isopropanol with equal volume, gently shaking and mixing, and standing for 15min. After that, the RNA was centrifuged at 12000rmp for 15min at 4℃and the supernatant was discarded, and the precipitated RNA was collected. To this, 1mL of pre-chilled 75% ethanol (DEPC water ready-to-use) was added, the centrifuge tube gently shaken, and the pellet rinsed. Centrifuging at 8000rmp at 4deg.C for 5min, discarding all supernatant, air drying at room temperature or vacuum drying for 5-10min, volatilizing ethanol, precipitating to become transparent, adding 40 μl DEPC water to dissolve RNA, and heating to 55-60deg.C to assist dissolution.
OD values were measured to quantify RNA concentration:
And (3) washing the sample obtained in the previous step with DEPC water for three times, zeroing the Nanodrop micro-spectrophotometer, and taking 1 mu L of sample liquid for measurement to obtain the concentration of RNA. The sample solution was then diluted to 3-4 ng/. Mu.L.
Reverse transcription of RNA:
A new tube was taken, DEPC water was added to the inside, and 2. Mu.g of RNA was then taken and prepared into a test system at a concentration of 12. Mu.L. Then heating at 65deg.C for 5min to make RNA become linear single strand, rapidly standing on ice, and cooling for 2min. Then Ncclease-FREE WATER and 4. Mu.L (4X) DNA MASTER Mix (gDNA reverse was added) were added to the system, and the reaction was allowed to stand at 37℃for 5 minutes to thereby remove the genomic DNA. Then, 4. Mu.L of q-PCR (5X) RT-Mix was added to prepare 20. Mu.L of a reaction system, the reaction mixture was gently mixed by shaking, and then reverse transcription was performed at a temperature of 37℃for 5min to 50℃for 5min to 98℃for 5min in order of time to obtain a cDNA sample solution.
q-PCR：
Preparing a q-PCR kit, pre-denaturing a template before Real-time PCR reaction, setting the temperature to 95 ℃ for 2min, and setting a q-PCR instrument according to the following parameters: pre-denaturation: 95 ℃,2 min- & gt denaturation: 95 ℃,15s→annealing/extension: 60 ℃,15-30 s- > repeat the two steps for a total of 40 cycles- > melting curve analysis: 95 ℃ for 15s;60 ℃ for 15s;95℃for 15s. After the reaction, the result is analyzed by using software provided by a q-PCR instrument.
In this example, representative dosage forms (liposome, chitosan nanoparticle) and representative derivatives (6) of 6-phosphogluconic acid were selected for testing, and it was determined that the target compound significantly increased the transcription level of insulin synthesis-related genes in islet alpha cells at a concentration of 10 μm (fig. 3). This example is not exhaustive of all possible formulations and derivatives, since, in theory, 6-phosphogluconate may enter the cells to release the active ingredient 6-phosphogluconate after optimization or derivatization of the formulation, thereby achieving the same desired effect.
EXAMPLE 7.6 preparation of Chitosan nanoparticles of phosphogluconate
Prescription of prescription
The operation is as follows: firstly, acetic acid (CH 3 COOH, > 99%) is diluted to 0.2% (volume fraction) acetic acid solution, 200 mu L of Tween 80 (tween-80) is added dropwise, 100mg of chitosan is weighed and dissolved into 100mL of acetic acid solution, and stirring is carried out for 30min until the chitosan is completely dissolved. The solution was filtered through a 0.22 μm filter membrane for further use. Taking 5mL of 6-phosphogluconic acid solution with the concentration of 10mg/mL, dropwise adding the solution into the chitosan solution at the rotation speed of 100rpm to form nano-microcapsules, and storing the nano-microcapsules in a refrigerator at the temperature of 4 ℃. The desired amount of nanoparticle suspension was mixed with PBS at ph6.8 and added to the cell culture dish for co-incubation.
EXAMPLE 8 preparation of liposomes of 6-phosphogluconate
Prescription of prescription
The operation is as follows: dioleoyl phosphatidylethanolamine (DOPE), cholesterol (Chol), (1, 2-dioleoxypropyl) trimethylammonium chloride (DOTAP) are weighed according to the prescription amount, 15mL of chloroform is added, and the mixture is dissolved in a 250mL round-neck flask, and is vibrated until the mixture is uniformly and completely dissolved. The chloroform solvent was distilled off under reduced pressure at 35℃to form a transparent film, and the residual solvent was removed by vacuum drying at room temperature. Adding 5-10mL of 6-phosphogluconate solution with the concentration of 10mg/mL, firstly swirling for 2min, hydrating at the constant temperature of 40 ℃ and carrying out ultrasound for 10min to obtain the milky semitransparent liposome solution. The resulting liposome solution was placed in a ultrafiltration tube (100 kDa MWCO) and centrifuged at 14000rpm at 4℃for 15min, and the concentrated liposome solution was taken and stored in a refrigerator at 4 ℃. The required amount of liposome solution was mixed with PBS at pH6.8 and added to the cell culture dish for co-incubation.
EXAMPLE 9 preparation of liposomes of 6-phosphogluconate
Liposome preparation of methyl 6-phosphogluconate
Prescription of prescription
The preparation method comprises weighing soybean lecithin, chol, DSPE-PEG 2000 in the recipe, and dissolving in 10mL chloroform; rotary evaporation at 40 ℃ to form liposome membrane; dissolving methyl 6-phosphogluconate in 10mL PBS with pH of 7.4, and adding into liposome membrane for hydration; ultrasonic treatment with cell pulverizer and film coating. The resulting liposome solution was placed in a ultrafiltration tube (100 kDa MWCO) and centrifuged at 14000rpm at 4℃for 15min, and the concentrated liposome solution was taken and stored in a refrigerator at 4 ℃. Experiments required amounts of liposome solution were mixed with PBS and added to a cell culture dish for co-incubation.
EXAMPLE 10 therapeutic Effect of 6-phosphogluconate pharmaceutical composition
Experimental apparatus and materials
1. Blood glucose test paper and blood glucose meter (American Gift company)
2. Female C57B 6/J mouse (Nanjing Ji Cui Ji Yi kang Co., ltd.)
3. Streptozotocin (sigma company of united states)
4. Hypersensitive insulin kit (company ALPCO U.S.)
5. Insulin antibodies (DAKO Co., USA)
6. Glucagon antibody (company ABCAM U.S.A.)
6. Other reagents required for immunofluorescence experiments are purchased from Shanghai Biotechnology Co
Construction of type I diabetes mouse model
C57B6/J female mice at 8 weeks of age were given a single intraperitoneal injection of 200mg/kg streptozotocin solution. After 5 days of injection, the random blood glucose levels of the mice were measured, and the mice with blood glucose levels higher than 20mmol/L were selected and randomly divided into two groups for the subsequent experiments.
Validation of 6-phosphogluconate derivatives on treatment effect of type I diabetes
Mice model of type I diabetes were given 200mg/kg of methyl 6-phosphogluconate (Compound 1-1) or physiological saline, once each of the early eight and late eight points daily. Mice were measured once daily for random blood glucose, fasted for 16 hours before the end of the experiment, and small amounts of peripheral blood were taken from the mice via the tail vein. Compared to the saline group, methyl 6-phosphogluconate treatment significantly reduced the random blood glucose levels in type i diabetic mice (fig. 4).
The regeneration of islet beta cells of type i diabetic mice was measured by immunofluorescence experiments. Compared to the saline group, methyl 6-phosphogluconate treatment increased islet beta cell number, leading to regeneration (fig. 5).
Validation of 6-phosphogluconic acid derivatives to improve insulin deficiency
The concentration of insulin in the peripheral blood of the mice was measured using a hypersensitive insulin kit. Compared to the saline group, methyl 6-phosphogluconate treatment significantly increased fasting insulin concentrations in type i diabetic mice (fig. 6).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (1)

  1. The use of 6-phosphogluconic acid and derivatives thereof, or pharmaceutically acceptable salts thereof, for the manufacture of a medicament for the prevention or treatment of a disorder of glucose metabolism, characterized in that the disorder of glucose metabolism is diabetes;
    the 6-phosphogluconate or the pharmaceutically acceptable salt thereof is modified by a drug delivery carrier which can penetrate cell membranes;
    The structure of the 6-phosphogluconate derivative is shown as a formula (I):
    In the method, in the process of the invention,
    R 1 is selected from alkyl groups with less than 10 carbon atoms, and R 2R3 is a hydrogen atom;
    or the 6-phosphogluconate derivative has a structure shown as any one of 20-1 and 23-1
    。
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